Viruses in the flavivirus family populate all three of NIAID's Category A, B, and C lists of viruses. Vaccines are needed for multiple flavivirus diseases, most notably dengue, and detailed knowledge of how flaviviruses interact with the innate immune system is critical for vaccine development. To address this need, we have developed a system to produce flavivirus virus-like particles (VLPs). VLPs are packaged, gene-deleted flaviviruses capable of initiating a replication cycle in cells in culture. VLPs are identical to normal flaviviruses in many aspects of infection, namely: receptor binding, uptake, RNA release, and RNA and nonstructural protein synthesis. However, VLP-infected cells do not produce functional virus capable of spreading to other cells. In addition to VLPs, we have produced a second type of flavivirus particle named RepliVAX. RepliVAX displays the non-spreading properties of VLPs, but in addition to encoding the viral products made by VLPs, RepliVAX also produces a sub-viral particle (SVP) with demonstrated capacity to induce antiviral immunity in man. RepliVAX is remarkably potent in mice and is being developed as a vaccine. Mice inoculated in the footpad with VLPs produce high levels of IFNa, in contrast to mice inoculated with UV-inactivated VLPs, which do not produce any IFNa. Draining lymph nodes (LN) harvested from VLP- inoculated mice contain WNV antigen and genome, and high levels of IFNa mRNA. These in vivo results suggest that VLPs are targeted to the draining LN where they induce IFNa synthesis. In vitro, VLP (and WNV) infection of cell lines derived from non-immune system cells induces production of a different type I IFN subtype (IFN[unreadable]), and induction is dependent on viral replication. IFN induction by WNV infection of human monocyte-derived dendritic cells (mDCs) is also dependent on viral replication, but the IFN produced by mDCs is of the IFNa subtype. On the other hand, human plasmacytoid DCs (pDCs), induce high levels of IFNa in response to exposure to live or inactivated WNV. Intriguingly, WNV does not productively infect pDCs. We hypothesize that WNV infects DCs (likely Langerhans cells) in the periphery, leading to their migration to the LN where they activate pDCs and recruit T and B cells. Further, we hypothesize that effective engagement of DC-centered innate signaling pathways contributes to the potency of our RepliVAX vaccine. To address these hypotheses, we will identify the precise nature of RepliVAX-infected cells in LN, determine if these cells (or neighboring cells) are responsible for the IFN production, and examine the effect of activation of these cells on the immune response to RepliVAX administration. These studies will provide important insights into links between innate and adaptive immunity that lead to the production of protective immunity to flavivirus infection and vaccination.